| Respiratory syncytial virus(RSV) is the major cause of pathogen of infection lower respiratory tract of infants. Acute bronchiolitis and pneumonia even leading death are happened in early infancy after infection. No safe and effective vaccine is yet available for respiratory syncytial virus(RSV). The effections on immunological function of mice are analysed and evaluated in this research by immunization of Balb/c with truncated RSV G aa130-230 (including CX3C motif, GCx3c),GCTL (substituting a CTL epitope of RSV M protein for CX3C, GCTL) alone or and adding mucosal adjuvant.GCx3C and GCTL large expressed by E.coli were purified by using affinity of His-Tag and Co2+. Immunization to Balb/c mice was arranged in 0,1,4 weeks after purified protein GCx3C and GCTL mixed with heat-labil enterotoxin, respectively, six groups were designed as follow:GCx3c group; GCTL group; GCx3C+LT group; GCTL+LT group; LT group; PBS group. When immunization ended, the quality indexes of immunization, such as IgG, IgA, IgG2a/IgGl, sIgA, histamine, eosinophil and lymphcytes proliferation, CTL responses ect., were measured by ELISA, HE and MTT staining. Balb/c mice were challenged with 107 pfu/ml RSV lasting 3 days post immunization. Mice were killed after challenge, and eosinophilia, histamine were detected. Meanwhil, lung tissue slices of mice were prepared. The results were analysed by SPSS16.0.GCx3C and GCTL were expressed with a great quantity by E. coli in inclusion body which were more than 40 percent of the total proteins, induced in 37℃. At last, purity more than 95% of GCx3c and GCTL were got, and the target proteins were confirmed by the Western blotting.The results of immunity analysises:Serum IgG after second immunization, GCTL,GCTL+LT groups'antibody titers were higher than Gcx3C+LT,GCx3C groups with significant differences (P<0.05), and four groups ahead were higher than LT,PBS groups with significant differences (P<0.05); Serum IgA after second immunization, GCTL,GCTL+LT groups'antibody titers were higher than GCx3C+LT,GCX3C groups with significant differences (P<0.05), and four groups ahead were higher than LT,PBS groups with significant differences (P<0.05); And in sIgA, two groups with LT got higher antibody titers than Gcx3c,GCTL,LT,PBS groups with significant differences (P<0.05); In examination of serum IgG2a/IgGl, GCTL group substituting RSV M protein'CTL epitope for CX3C got better balance function on Th1/Th2 than GCTL+LT,GCX3c+LT,GCx3c groups (P<0.05), and physiological functions of LT which could adjust immunal polarization in coordination of Th1/Th2 were detected; In vitro lymphcyte proliferation, after stimulation with antigens, proliferation only happened in GCx3c and GCTL groups, contrary phenomena emerged in control group and adjuvant group, and vaccine groups had a larger proliferation than control group and adjuvant group with significant differences (P<0.05); The eosinophilia number in mice'blood of Gcx3C,Gcx3c+LT groups were higher than GCTL,GCTL+LT,PBS groups with significant differences (P<0.05); Histamine of Gcx3c group was higher than GCTL group and PBS group with significant differences(P<0.05); In the tissue slice results, there were no significant inflamations in protein GCTL groups, but obvious inflamations happened in protein Gcx3c groups; In the RSV-specific CTL responses, GCTL group induced significantly RSV-specific CTL responses, and Gcx3c group also induced CTL responses against RSV.The results of RSV challenge:The eosinophilia number in mice'blood of Gcx3c Gcx3C+LT groups were higher than GCTL,GCTL+LT,PBS groups with significant differences (P<0.05) after challenge; In the results of histamine, GCx3c groups was higher than GCTL groups with significant differences(P<0.05); No marked pathological changes were observed in GCTL group, but in GCx3c,GCx3C+LT and PBS groups.The recombinant proteins Gcx3C,GCTL and LT were successfully expressed and purified, the effections of immunes on immunogenicity after immunization and protection after challenge of mice were detected. The results of experiments demonstrated that vaccine mixed with LT could elevate the quantity of specific sIgA. The reconstitution to aim directly at GCx3C is successful and effective, the down regulation was happened to eosinophilia of the respiratory tract of mice, and the RSV-specific CTL responses were enhanced by GCTL protein. Parts experiments post challenge also reflected desired actions of GCTL protein in protection from one side. All these results indicated that the GCTL could decrease more-severe lung diseases during the subsequent RSV infection. The experiments on mice of RSV G protein subunit vaccine provid a good strategy in development of new generation of RSV vaccine. |
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